The present invention relates in general to methods and pharmaceutical compositions for treating inflammation, and more particularly, to a method and pharmaceutical composition for downregulating adhesion and migration of lymphocytes and, specifically, to the administration of ultra-low dosages of interferon gamma (IFN-γ) to treat a variety of immunopathological states which are accompanied by inflammation, such as, but not limited to, autoimmune disease, allergy, inflammation and graft rejection.
Many lymphocyte-mediated immune processes require adhesion of lymphocytes to the ECM. Such adhesion is critical, for example, for B and T lymphocyte extravasation and migration during differentiation, activation, proliferation and effector function during both normal and pathological immune processes. As such, selectively interfering with adhesion and migration of lymphocytes can be directly and effectively applied towards treatment of disorders involving lymphocyte-mediated pathogenesis.
Specific Immunity:
The specific arm of the immune system employs B and T lymphocytes to recognize and eliminate foreign antigens, such as foreign microorganisms, foreign substances or allogeneic cells, while remaining unresponsive to self antigens. Targeting of specific foreign antigens and elimination of foreign bodies displaying these is mediated via B and T lymphocytes which, following somatic gene rearrangement and clonal selection, respectively express antibodies specific to a particular foreign antigen or T cell receptors (TCRs) specific to a particular major histocompatibility complex (MHC)-foreign peptide complex. The humoral arm of the immune system thus employs antibodies to eliminate, for example, bacteria and foreign substances, whereas the cellular arm of the immune system employs T lymphocytes to eliminate, for example, virus- or parasite-infected cells, cells expressing mutated self-antigens, such as tumor cells or allogeneic cells.
Trafficking of Lymphocytes During Immune Responses
During the processes of immune surveillance and specific immune responses, lymphocytes extravasate and migrate to secondary lymphoid organs, such as lymph nodes (LNs), for antigen-specific activation after which these migrate to the sites of inflammation for execution of effector functions against foreign pathogens. Extravasation of lymphocytes to such sites occurs through specialized high endothelial venules (HEV) via recognition of organ-specific adhesion molecules by counter-receptors, such as integrins, expressed on lymphocytes (Bradley, L., and Watson, S. R. Curr. Opin. Immunol. 1996, 8:312; Imhof, B. A., and Dunon, D. Adv. Immunol. 1995, 58:345). These processes involve multiple adhesion steps regulated via a combinatorial series of molecular events.
Continuous recirculation of lymphocytes to organized lymphoid tissue or to sites of antigen-presentation, such as LNs, is necessary for the development of primary immune responses to foreign antigen by lymphocytes and such recirculation from one anatomic compartment to another is critically dependent on adhesive interactions of lymphocytes with endothelium and with the extracellular matrix (ECM) for extravasation and migration within tissues, such as sites of inflammation, respectively.
The process of B cell development also involves distinct trafficking patterns and thus is also critically dependent on regulation of adhesion of B lymphocytes to endothelium and to ECM. Immature B cells initially differentiate in the bone marrow from which they exit to the periphery and selectively migrate into the spleen to complete their maturation. While in transit to the spleen, specific mechanisms prevent the entry into and/or retention of immature B cells in sites of antigen-presentation, such as sites of inflammation or secondary lymphoid organs, such as LNs. This is due to the fact that maturation of B cells occurs in anatomic compartments in which auto-antigens induce the clonal deletion of autoreactive immature B cells. Thus, such cells are prevented from entering sites of antigen-presentation so as to prevent deletion of foreign antigen-reactive clones.
The specific mechanisms of lymphocyte trafficking during immune responses are best characterized for T cells, as detailed below.
Non-activated T lymphocytes traffic through the T cell areas of secondary lymphoid organs, such as LNs, where they encounter antigen presented by dendritic cells (DCs), thereby triggering activation of antigen-specific T cells (Butcher, E. C., and Picker, L. J. Science 1996, 272:60; Banchereau, J., and Steinman, R. M. Nature 1998, 392:245). In the presence of polarizing cytokines, activated T lymphocytes acquire effector functions and differentiate into Th1 or Th2 subtypes displaying characteristic cytokine production profiles and mediating pro-inflammatory and allergic types of responses, respectively (Abbas, A. K. et al., Nature 1996, 383:787; O'Garra, A. Immunity 1998, 8:275). Such differentiated T cells downregulate LN homing receptors and upregulate specific tissue homing receptors (Xie, H.et al., J. Exp. Med. 1999, 189:1765; Potsch, C. et al., Eur. J. Immunol. 1999, 29:3562; Campbell, J. J., and Butcher, E. C. Curr. Opin. Immunol. 2000, 12:336; Sallusto, F. et al., Annu. Rev. Immunol. 2000, 18:593) resulting in Th1 and Th2 cells exhibiting distinct migratory profiles in vivo (Austrup, F. et al., Nature 1997, 385:81; Randolph, D. A. et al., Science 1999, 286:2159).
During immune responses to pathogenic insult, foreign antigens present in affected tissues are taken up by antigen-presenting DCs which migrate to sites of antigen-presentation, such as LNs. Meanwhile, components of the non-specific cellular immune system, such as neutrophils and other granulocytes, initiate inflammation by, for example, releasing pro-inflammatory molecules, such as chemokines and cytokines which activate local endothelial cells to upregulate expression of lymphocyte-specific adhesion molecules, such as immunoglobulin superfamily ligands. In sites of antigen-presentation, such as LNs, DCs presenting foreign antigens activate antigen-specific lymphocytes. Activated lymphocytes then upregulate adhesion molecules, such as integrins specific for activated endothelial cells (ECs), exit to the circulation and extravasate at sites of inflammation.
In the circulation, transient interactions between lymphocytes and endothelium enable lymphocyte tethering and rolling along the endothelial wall. During this rolling phase, lymphocytes are activated by intracellular signals generated by engaged adhesion molecules, such as selectins and chemokine receptors, which transduce signals promoting firm adhesion between integrin molecules, expressed on lymphocytes, and their immunoglobulin superfamily ligands expressed on the endothelial wall. Such adherent lymphocytes then extravasate through the intercellular margins of the endothelium and migrate, via adhesion-disruption cycles, through the ECM to reach foreign-antigen containing inflamed tissue wherein effector functions are performed (Butcher, E. C. Cell 1991, 67:1033; Shimizu, Y., W. et al., Immunol. Today 1992, 13:106; Mackay, C. R., and B. A. Imhof. Immunol. Today 1993, 14:99; Schall, T. J., and K. B. Bacon. Curr. Opin. Immunol. 1994, 6:865; Hogg, N., and C. Berlin. Immunol. Today 1995, 16:327; Imhof, B. A., and D. Dunon. Adv. Immunol. 1995, 58:345; Butcher, E. C., and J. Picker. Science 1996, 272:60; Springer, T. Annu. Rev. Physiol. 1995, 57:827).
The trafficking signals directing activated effector T cells to peripheral tissues are organ-specific and are distinct for the different subgroups of T cells. For example, naïve T cells express CD62 ligand (CD62L) and CC chemokine receptor 7 (CCR7), which are required for HEV extravasation (Springer, T. A. Cell 1994, 76:301; Cyster, J. G. Science 1999, 286:2098; Stein, J. V. et al., J. Exp. Med. 2000, 191:61; Forster, R. et al., Cell 1999, 99:23). Upon foreign antigen encounter at sites of inflammation, activated lymphocytes release cytokines and chemokines which amplify the inflammatory cascade thereby increasing vascular and ECM permeability thus facilitating infiltration of effectors to inflamed sites of foreign pathogen infiltration. Such inflammatory processes normally result in a certain amount of tissue destruction which, when appropriately regulated, is tolerated by the body as a reversible consequence of optimal immune responses.
Thus, adhesion of lymphocytes to endothelium and the ECM is critical to extravasation and migration during processes such as lymphocyte maturation, antigen-specific activation and effector responses against foreign antigens at sites of inflammation.
Lymphocyte-Mediated Disorders:
Although lymphocyte-mediated immune processes normally serve to allow the immune system to fight foreign pathogens, in certain contexts such immune processes can lead to pathological states referred to as hypersensitivity diseases. Alternatively, transplantation of foreign cells, tissues or organs or surgically implanted prosthetic devices can also elicit undesirable immune responses against such implants in recipients subjected to such therapeutic interventions. In such diseases, lymphocytic infiltration into tissues and the consequences thereof, such as cytokine production, significantly contribute to undesirable sequelae, such as acute tissue injury, resulting from uncontrolled inflammation.
Such uncontrolled inflammation may be a result of antibody-mediated diseases such as allergy (immediate hypersensitivity) or immune complex deposition. Alternatively, uncontrolled inflammation may be a result of T lymphocyte-mediated diseases such as contact dermatitis and drug eruptions (delayed hypersensitivity). Infection or cancer may also result in uncontrolled inflammation.
Importantly, deregulated immune responses may specifically target self-antigens, either idiopathically, as a result of cross-reactivity with foreign antigen or, alternately, immunity directed against foreign antigens may cause damage to specific tissues to which such foreign antigens have a specific affinity.
Such hypersensitivity diseases have been implicated in an extremely broad range of autoimmune diseases including systemic, cutaneous, rheumatoid, cardiovascular, gastrointestinal, hepatic, reproductive, glandular, neurological, muscular, nephric and connective tissue autoimmune diseases, as described in the Preferred Embodiments section.
Role of Lymphocytes in Transplantation Failure:
Immune reactions may also be deleterious in the context of therapeutic tissue transplantation. For example, T lymphocytes are responsible for transplantation-related immunopathologies such as allograft rejection (Krensky A. et al., N Engl J Med. 1990 Feb. 22; 322 (8):510) and GVHD (Theobald, M. Transfus Sci 1994 September; 15 (3):189) which are the major causes of transplantation failure. In allograft rejection and in GVHD, respectively, immune responses are mediated by T cells of the host or of the donor, respectively, which extravasate and migrate into donor and host tissues, respectively, to induce disease. In the case of graft rejection, host T lymphocytes also migrate into secondary lymphoid organs where they are primed against graft-derived allogeneic antigens.
Prior Art Therapy Using IFN-γ:
Satisfactory IFN-γ-based therapies for many diseases involving inflammation, such as autoimmune diseases, allergic diseases, transplantation failure and cancer, as described above, have yet to be developed. Prior art approaches of treating inflammation-related disorders using IFN-γ have employed administration of high levels of this cytokine, as described below.
Animal studies: The use of high levels of IFN-γ to regulate inflammatory processes has been attempted in animal models of autoimmune disease. In a murine asthma model, treatment of tracheal eosinophil and CD4+ T cell infiltration was achieved using intraperitoneal administration of high levels of IFN-γ, at doses of 24,000 and 240,000 units per kilogram body weight administered per day, respectively, in response to ovalbumin (OVA) inhalation (Iwamoto, I. et al., J. Exp. Med. 1993, 177:573).
Human studies: Since high levels of IFN-γ were shown to be of potential therapeutic benefit in animal studies, such as the one described above, therapy employing high levels of IFN-γ has been attempted in human clinical trials for treatment of autoimmune disease. Such treatments, however, have been found to produce unacceptably severe side-effects in patients.
For example, in a clinical trial of long-term treatment of the inflammatory disease idiopathic pulmonary fibrosis, high levels of IFN-γ (33,000 units per kilogram body weight administered three times per week) were required in order to provide substantial therapeutic effect (Ziesche, R. et al., N. Engl. J. Med. 1999, 341:1264). All patients treated in this study, however, developed fever and chills and one-third experienced significant bone and muscle pain as a result of high-dose IFN-γ administration.
In an open pilot study employing IFN-γ therapy for treatment of another inflammatory disease, Crohn's disease, administration of high levels of IFN-γ (15,000 units/kg administered three times per week) led to a substantial decrease in Crohn's disease activity index in most patients, however, most patients did not finish the 12-week treatment course due to suboptimized dosages and an unacceptable incidence of side-effects (Debinski H. et al., Ital J Gastroenterol Hepatol 1997 October; 29(5):403). It should be noted that although the title of the latter publication refers to the employed dose of 15,000 units per kilogram body weight IFN-γ as being a “low” dose, the method of the present invention, as described in the Examples section, below, employs ultra-low doses of IFN-γ being at least two orders of magnitude lower than such doses.
The undesirable side-effects of high doses of IFN-γ were also demonstrated in a human clinical trial for treatment of metastatic renal cell carcinoma in which patients were treated with high levels of IFN-γ (107 units IFN-γ per m2 per day for 5 days, administered every two weeks for four weeks, followed by repeated administration of such dose, three times a week for 2 weeks. A dose of 107 units/m2 corresponds to over 100,000 units per kg body weight in an average 60 kg human) (Griebel, P. et al., Int. Immunol. 1999, 11:1139). These patients experienced major side-effects including fever and chills (75%), anorexia and fatigue (75%), nausea and vomiting (80%), leukopenia (38%) and abnormal liver function (25%).
Thus, as a result of the significant side-effects of high-dose IFN-γ treatment described above, this cytokine is not currently employed therapeutically. The deleterious effects of high-dose IFN-γ may result from its potentiation of the effects of other pro-inflammatory mediators and upregulation of surface expression of MHC class II in monocytes, macrophages and dendritic cells, as well as of adhesion molecules in endothelial and epithelial cells (Barnes, P. J., and Lim, S. Mol. Med. Today 1998, 4:452).
There is thus a widely recognized need for, and it would be highly advantageous to have, a method of treatment of disease with IFN-γ devoid of the above limitations.